Lysophospholipids constitute a class of membrane glycerophospholipids that are rapidly transported and metabolized by many eukaryotic cells ranging from yeast to humans. We have recently identified two yeast transporters for the major lysophospholipids found throughout the eukaryotic world, lyso- phosphatidylethanolamine (lyso-PtdEtn) and lyso-phosphatidylcholine (lyso-PtdCho). The yeast lyso-PtdEtn and lyso-PtdCho transporters are also the same molecules implicated in regulating the plasma membrane asymmetry of phosphatidylserine and phosphatidylethanolamine. The metazoan forms of these transporters play crucial roles in signaling the apoptotic status of target cells to phagocytes. Defects in the function of the mammalian transporters are linked to primary familial intrahepatic cholestasis and benign recurrent intrahepatic cholestasis in humans. Genetic and biochemical work in yeast reveals that additional lyso-PtdCho and lyso-PtdEtn transporters remain to be identified. The first specific aim of this proposal will focus on identifying the genes for the remaining yeast lysophospholipid transporters and characterizing them biochemically. The second specific aim will capitalize upon the powerful tools of yeast genetics to structurally define the mechanistic basis by which the yeast and mammalian transporters recognize their substrates and introduce them into the transport channel. Subsequent to uptake by yeast and mammalian cells, lyso-PtdCho and lyso-PtdEtn are metabolized to their diacyl counterparts, PtdCho and PtdEtn, by acyltransferase enzymes. Although the enzyme activities have been known for more than 40 years, our recent work in yeast provided the first definition of the genes executing these reactions. The yeast gene ALE1 is the founding member of a family represented in multiple eukaryotes that is involved in acylating lysophospholipids with di-unsaturated and polyunsaturated fatty acids. In the third specific aim we will use the power of yeast genetics to elucidate the biochemical activities and functions of the metazoan acyltransferases belonging to the ALE1-family. Genetic studies already reveal that lesions in Drosophila ALE1-family genes cause developmental defects and male sterility. Studies in humans reveal defects in ALE1-family genes cause developmental defects of the hands and skeleton and also produce male sterility. The biochemical studies in yeast will be complemented by detailed studies in mammalian cells examining the role of ALE1-family members as regulators of arachidonic acid mobilization for the synthesis of eicosanoids. In summary, the work in this proposal will define major genetic and biochemical aspects of lysophospholipid transport and metabolism that are intimately associated with fundamental processes of cell biology whose dysfunction leads to serious human disease. PUBLIC HEALTH RELEVANCE: The focus of this proposal is upon genes and enzymes involved in transport and metabolism of a class of lipids known as lysophospholipids. Dysfunction of genes involved in lysophospholipid transport is known to cause the liver diseases Progressive Familial Intrahepatic Cholestasis and Benign Recurrent Intrahepatic Cholestasis. Dysfunction of genes involved in the metabolism of lysophospholipids is linked to developmental abnormalities of the hands and sterility in humans. The goals of this project are to understand the mechanistic biochemistry of how lysophospholipid transport occurs and how the lipids are metabolized.